- Email: [email protected]
Direct Automated Sequencing of Single λ-Phage Plaques by Exponential Amplification Sequencing
460
NOTES & TIPS
dation. Several reports (8,9) have noted the problem of extensive proteolytic degradation when protein tyrosine phosphatase 1B is expressed in E. coli. This problem led several laboratories to express only the catalytic domain, which is more stable (8–10). Using the strategy described in this paper, we were able to express and purify full-length protein tyrosine phosphatase 1B (0.2 mg purified/liter of culture). In summary, the use of two affinity tags allows rapid and efficient removal of the contaminating degradation products common to many current fusion protein expression systems. It also allows the removal of the protease, thrombin in this study, from the final purified protein, avoiding potential nonspecific proteolysis of the target protein in subsequent studies. Acknowledgments. This work was supported by the John S. Dunn Research Foundation (Houston, TX), IRSC 8-0070278, and NCI Grant CA53617. We thank M. Risin, H. Saya, H. Takeshema, and P. Lee for their help.
REFERENCES 1. Smith, D. B., and Johnson, K. S. (1988) Gene 67, 31–40. 2. Guan, K., and Dixon, J. E. (1991) Anal. Biochem. 192, 262–267. 3. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 4. Cooper, J. A., and Howell, B. (1993) Cell 73, 1051–1054. 5. Bougeret, C., Rothhut, B., Jullien, P., Fischer, S., and Benarous, R. (1993) Oncogene 8, 1241–1247. 6. Budde, R. J. A., Ramdas, L., and Ke, S. (1993) Prep. Biochem. 23, 493–515. 7. Schmidt, T. G. M., and Skerra, A. (1993) Protein Eng. 6, 109– 122. 8. Guan, K., and Dixon, J. E. (1991) J. Biol. Chem. 266, 17026– 17030. 9. Hoppe, E., Berne, P. F., Stock, D., Rasmussen, J. S., Moller, N. P. H., Ullrich, A., and Huber, R. (1994) Eur. J. Biochem. 223, 1069–1077. 10. Barford, D., Flint, A. J., and Tonks, N. K. (1994) Science 263, 1397–1404.
Direct Automated Sequencing of Single l-Phage Plaques by Exponential Amplification Sequencing
Materials and Methods
David M. Hwang, Ruo-Xiang Wang, and Choong-Chin Liew1 Laboratory of Molecular Cardiology, Departments of Clinical Biochemistry and Medicine, The Centre for Cardiovascular Research, The Toronto Hospital, University of Toronto, Toronto, Ontario, Canada Received September 6, 1995
Linear amplification sequencing (1) involves cycling the components of a Sanger sequencing reaction 1 To whom correspondence should be addressed at Banting Institute, University of Toronto, 100 College St., Toronto, Ontario M5G 1L5, Canada. Fax: (416)-978-5650. e-mail: [email protected].
ANALYTICAL BIOCHEMISTRY
through multiple denaturation, annealing, and extension steps, in the presence of a single, specific oligonucleotide primer and a thermostable DNA polymerase. Because only one primer is used, cycling of the reactions results in linear amplification (as opposed to exponential amplification in PCR2) of the sequencing ladder, reducing the amount of template required for the sequencing reaction by a factor of the number of cycles used (typically up to 25- to 30-fold). We have previously reported the application of PCR and cycle sequencing methodologies to the large-scale sequencing of human heart cDNA libraries (2–4). Our protocol involved PCR amplification of cDNA inserts from l-phage plaque suspensions, followed by cycle sequencing of the PCR products using a nested, fluorescein-labeled primer. Recently, we explored the possibility of sequencing inserts directly from phage plaques. While linear amplification sequencing of single l plaques had been described using manual sequencing techniques (5), this approach has not been successfully generalized to the automated sequencing technologies used in most largescale sequencing projects, probably due to the currently lower sensitivity of these technologies. Other attempts to sequence very small quantities of template included coupled amplification and sequencing (6) and semiexponential cycle sequencing (SECS) (7). However, because both techniques are two-step processes, involving a PCR step followed by cycle sequencing of the PCR product, they have not yet been, in practice, particularly advantageous over more traditional template isolation and sequencing protocols currently used by large-scale sequencing projects. We therefore developed a method for single-step sequencing directly from individual l-phage plaques by exponential amplification (EAS). In this report, we present results of EAS performed on single plaques from a directionally cloned human fetal heart cDNA library, and we demonstrate its potential utility to large-scale sequencing projects.
Exponential amplification sequencing. A human fetal heart (10–12 weeks) l ZAP-Express cDNA library was constructed and plated using XL1-Blue as a host strain, using isopropyl b-D-thiogalactoside/X-gal color selection. Individual plaques were picked with glass Pasteur pipets and each was eluted in 100 ml SM buffer, as previously described (2). EAS reactions were conducted essentially according to the manufacturer’s recommended protocol using the Taq Cyclist DNA Sequenc2 Abbreviations used: PCR, polymerase chain reaction; SECS, semiexponential cycle sequencing; EAS, exponential amplification sequencing; EST, expressed sequence tag.
231, 460–463 (1995)
0003-2697/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ m4314$$nat
10-13-95 16:04:50
abnt
AP-Anal Bio
NOTES & TIPS
ing Kit (Stratagene). Beyond the buffer and nucleotide components provided by the kit (10 mM Tris–HCl, pH 8.8, 50 mM KCl, 0.001% gelatin, 4 mM MgCl2 , 2 mM dATP, 5 mM dCTP, 5 mM dGTP, 5 mM dTTP), the EAS master mix contained 10 pmol fluorescein-labeled modified T3 primer (5*-fluorescein-GAAATTAACCCTCACTAAAGGG-3*), 10 pmol unlabeled modified T7 primer (5*-CCAGTGAATTGTAATACGACTCACTATCGGGCG-3*), an additional 312.5 pmol each dNTP (1 ml 1 312.5 mM), 2 ml dimethyl sulfoxide, 1 ml phage suspension, and 5 U Taq, in a final total volume of 30 ml. Master mix (7 ml) was aliquoted into each of four tubes containing, respectively, 3 ml ddATP (600 mM), ddCTP (600 mM), ddGTP (100 mM), or ddTTP (1000 mM) (manufacturer’s concentrations). Reactions were overlaid with mineral oil and heated at 947C for 2 min, followed by 35 cycles at 947C, 30 s; 507C, 25 s; and 727C, 1 min 20 s; followed by a 5-min extension at 727C. Reactions were stopped by addition of 5 ml stop solution (95% formamide, 20 mM EDTA, 10 mg/ml blue dextran). Prior to electrophoresis, samples were heated to 957C. Samples were electrophoresed through a 6% acrylamide gel, using the Pharmacia A.L.F. DNA Sequencer. Data handling. Sequence comparisons were performed using the BLAST server at the National Center for Biotechnology Information (8,9) and the HIBIO package (Hitachi). Results and Discussion Table 1 summarizes the results of direct sequencing from individual plaques from a human fetal heart l ZAP-Express cDNA library by EAS. Sequences ranged in length from 142 to 503 bp, with the average length being approximately 296 bp. Sequencing reactions which were conducted in the absence of additional dNTP or unlabeled primer, or both, failed to generate detectable sequences (data not shown). Of 49 partial cDNA sequences, or expressed sequence tags (ESTs) obtained, 15 demonstrated no homology to known genes, while 3 exhibited similarity to other human ESTs, though not to any other known genes (Table 1). The remaining 31 were similar to previously known genes, including 2 novel human homologs of genes characterized in the cow. Of the 31 known ESTs, 7 represented mitochondrial transcripts (23%), while another 7 represented ribosomal proteins (23%). Assessment of sequencing accuracy by comparison of EST sequences with known gene sequences found that most known ESTs demonstrated between 98 and 100% identity to known gene sequences (Table 1), suggesting a high level of accuracy. The 296-bp average sequence length reported here exceeds the 200 bp previously reported for manual linear amplification sequencing from a single l-phage
/ m4314$$nat
10-13-95 16:04:50
abnt
461
plaque (5). Moreover, while the previous protocol used 80% of the phage plaque suspension, the EAS protocol described in this report used only 1% (i.e., 1 of 100 ml) of phage suspensions. This represents at least an 80fold decrease in the amount of template required for sequence generation or, conversely, an 80-fold increase in sensitivity over conventional cycle sequencing. Assuming the average l-phage plaque to contain 106 – 107 phage particles (10), and assuming each particle to represent one copy of the phage genome, this represents only 104 –105 copies, an unprecedented sub-attomole quantity of template required to generate quality sequence. Titering of phage suspensions demonstrated as few as 4 1 103 pfu required for sequence generation and detection (data not shown). Assuming similar requirements for template quantity in direct sequencing of genomic DNA, this method represents a requirement of only 0.02–0.4 mg of genomic DNA in order to sequence single-copy genes and would theoretically permit direct sequencing from genomic DNA isolated from less than a single drop of blood. Given the currently lower sensitivity of automated sequencing than manual techniques, it is reasonable to assume that the template requirements for manual sequencing protocols should be even lower. The rationale underlying the EAS technique is simple and similar to that of SECS (7). Standard cycle sequencing reactions (containing an excess of a fluorescein-labeled sequencing primer) were performed in the presence of additional nucleotides (dNTPs) and of an additional, unlabeled PCR primer, opposite in sense to the sequencing primer. The presence of additional dNTPs decreases the frequency of early termination by ddNTP and increases the probability of full-length extension from one primer to the annealing site of the other primer. Thus, in each cycle, a proportion of primer extensions from each primer would extend the full length of the fragment to be sequenced, thereby allowing for exponential amplification of the template. Concurrently, termination of the remainder of primer extensions by ddNTP leads to generation of a sequencing ladder. In early stages of cycling, when relatively more dNTP is available, proportionally more exponential amplification of the template occurs. In later stages, however, as dNTP becomes depleted, and as the ratio of dNTP/ ddNTP decreases, proportionally more ddNTP is incorporated, generating shorter fragments, thereby progressively diminishing the rate of exponential amplification, while progressively increasing the rate of linear amplification sequencing. In essence, EAS merges the concepts of PCR and linear amplification sequencing into a single step. It should be noted at this point that although sequencing ladders would be generated from both primers, only the ladder originating from the labeled primer would be visualized. To date, EAS has been successfully applied only to
AP-Anal Bio
462
NOTES & TIPS TABLE 1
Partial Sequences of cDNA Clones from a Human Fetal Heart cDNA Library by EAS GB/EMBL/DDBJ Accession Nos. R56917–R56964
Clone
Sequence length (bp)
Putative identity
EAS-J2362 EAS-J2376 EAS-J2390 EAS-J2396 EAS-J2403 EAS-J2420 EAS-J2434 EAS-J2491 EAS-J2499 EAS-J2517 EAS-J2765 EAS-J3574 EAS-J3596 EAS-J3606 EAS-J3608 EAS-J2782 EAS-J2757 EAS-J2346 EAS-J2374 EAS-J2356 EAS-J2363 EAS-J2767 EAS-J2525 EAS-J3590 EAS-J3611 EAS-J2383 EAS-J1323 EAS-J3610 EAS-J2436 EAS-J2500 EAS-J2364 EAS-J2768 EAS-J1319 EAS-J1322 EAS-J2392 EAS-J2424 EAS-J2774 EAS-J3582 EAS-J3613 EAS-J2775 EAS-J2412 EAS-J2761 EAS-J1324 EAS-J3588 EAS-J3609 EAS-J3594 EAS-J2780 EAS-J2400 EAS-J2433
142 307 400 328 267 279 211 295 266 262 338 165 241 336 299 503 412 322 323 360 330 206 317 227 233 337 333 331 188 246 222 207 302 337 412 301 291 208 312 259 243 351 450 165 287 358 334 407 277
Novel Novel Novel Novel Novel Novel Novel Novel Novel Novel Novel Novel Novel Novel Novel EST hbc2678 EST HSB92G052 EST IB287, IB2438 a-Globin Alu repetitive element Alu repetitive element Cardiac a-myosin heavy chain CCAAT/enhancer-binding protein d Chloride channel (cow) Chorionic gonadotropin Cytochrome c oxidase subunit VIIc EDZ protein Elongation factor 1-g Endozepine F1F0 ATPase synthase domain g subunit (cow) Ki nuclear autoantigen Kinesin heavy chain Mitochondrial Mitochondrial Mitochondrial Mitochondrial Mitochondrial Mitochondrial Mitochondrial Myosin light chain Open reading frame Ribosomal protein Ribosomal protein L3 Ribosomal protein L23 Ribosomal protein L27a Ribosomal protein L31 Ribosomal protein L32 Ribosomal protein S11 Ubiquinol cytochrome c reductase
% match
Length of match
94 99 96 96
246 304 184 308
98 97 74 98 98 98 99 97 88 98 99 96 99 99 99 98 98 99 99 98 98 98 98 98 100 99 98 96
206 316 91 233 288 301 331 188 238 221 206 302 319 412 300 291 208 298 258 243 342 420 163 287 358 333 381 132
Note. All matches listed are with human sequences, unless otherwise denoted in parentheses.
the sequencing of relatively short cDNA inserts (up to 1.1 kb; data not shown), likely because longer templates have a lower probability of full-length extension at the dNTP/ddNTP ratio used in this report. It is therefore expected that the dNTP/ddNTP ratio will be
/ m4314$$nat
10-13-95 16:04:50
abnt
a critical factor in optimizing EAS conditions to permit sufficient amplification at longer insert lengths. Nevertheless, the implications and applications of EAS are potentially numerous. Generation of highquality sequences directly from phage plaques or from
AP-Anal Bio
NOTES & TIPS
single bacterial colonies would reduce time and costs required for template isolation prior to sequencing. EAS may therefore be of great interest to large-scale sequencing projects making use of dye-primer-based sequencing chemistries, for example, in the rapid sequencing of size-selected shotgun libraries. Moreover, the ability to sequence from very small quantities of genomic DNA for rapid genotype analysis might be of interest to the field of molecular diagnostics. Acknowledgments. We acknowledge the excellent assistance of H. Ma and X.-G. Zhao. This work was supported in part by the Medical Research Council of Canada and by the Heart and Stroke Foundation of Ontario. D.M.H. is supported by a Medical Research Council of Canada Studentship.
463
2. Liew, C. C. (1993) J. Mol. Cell. Cardiol. 25, 891–894. 3. Liew, C. C., Hwang, D. M., Fung, Y. W., Laurenssen, C., Cukerman, E., Tsui, S., and Lee, C. Y. (1994) Proc. Natl. Acad. Sci. USA 91, 10645–10649. 4. Hwang, D. M., Hwang, W. S., and Liew, C. C. (1994) J. Mol. Cell. Cardiol. 26, 1329–1333. 5. Krishnan, B. R., Blakesley, R. W., and Berg, D. E. (1991) Nucleic Acids Res. 19, 1153. 6. Ruano, G., and Kidd, K. K. (1991) Proc. Natl. Acad. Sci. USA 88, 2815–2819. 7. Sarkar, G., and Bolander, M. E. (1995) Nucleic Acids Res. 23, 1269–1270. 8. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403–410. 9. Gish, W., and States, D. J. (1993) Nature Genet. 3, 266–272.
REFERENCES 1. Saluz, H., and Jost, J. P. (1989) Proc. Natl. Acad. Sci. USA 86, 2602–2606.
/ m4314$$nat
10-13-95 16:04:50
abnt
10. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, pp. 2–63, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
AP-Anal Bio
NOTES & TIPS
dation. Several reports (8,9) have noted the problem of extensive proteolytic degradation when protein tyrosine phosphatase 1B is expressed in E. coli. This problem led several laboratories to express only the catalytic domain, which is more stable (8–10). Using the strategy described in this paper, we were able to express and purify full-length protein tyrosine phosphatase 1B (0.2 mg purified/liter of culture). In summary, the use of two affinity tags allows rapid and efficient removal of the contaminating degradation products common to many current fusion protein expression systems. It also allows the removal of the protease, thrombin in this study, from the final purified protein, avoiding potential nonspecific proteolysis of the target protein in subsequent studies. Acknowledgments. This work was supported by the John S. Dunn Research Foundation (Houston, TX), IRSC 8-0070278, and NCI Grant CA53617. We thank M. Risin, H. Saya, H. Takeshema, and P. Lee for their help.
REFERENCES 1. Smith, D. B., and Johnson, K. S. (1988) Gene 67, 31–40. 2. Guan, K., and Dixon, J. E. (1991) Anal. Biochem. 192, 262–267. 3. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 4. Cooper, J. A., and Howell, B. (1993) Cell 73, 1051–1054. 5. Bougeret, C., Rothhut, B., Jullien, P., Fischer, S., and Benarous, R. (1993) Oncogene 8, 1241–1247. 6. Budde, R. J. A., Ramdas, L., and Ke, S. (1993) Prep. Biochem. 23, 493–515. 7. Schmidt, T. G. M., and Skerra, A. (1993) Protein Eng. 6, 109– 122. 8. Guan, K., and Dixon, J. E. (1991) J. Biol. Chem. 266, 17026– 17030. 9. Hoppe, E., Berne, P. F., Stock, D., Rasmussen, J. S., Moller, N. P. H., Ullrich, A., and Huber, R. (1994) Eur. J. Biochem. 223, 1069–1077. 10. Barford, D., Flint, A. J., and Tonks, N. K. (1994) Science 263, 1397–1404.
Direct Automated Sequencing of Single l-Phage Plaques by Exponential Amplification Sequencing
Materials and Methods
David M. Hwang, Ruo-Xiang Wang, and Choong-Chin Liew1 Laboratory of Molecular Cardiology, Departments of Clinical Biochemistry and Medicine, The Centre for Cardiovascular Research, The Toronto Hospital, University of Toronto, Toronto, Ontario, Canada Received September 6, 1995
Linear amplification sequencing (1) involves cycling the components of a Sanger sequencing reaction 1 To whom correspondence should be addressed at Banting Institute, University of Toronto, 100 College St., Toronto, Ontario M5G 1L5, Canada. Fax: (416)-978-5650. e-mail: [email protected].
ANALYTICAL BIOCHEMISTRY
through multiple denaturation, annealing, and extension steps, in the presence of a single, specific oligonucleotide primer and a thermostable DNA polymerase. Because only one primer is used, cycling of the reactions results in linear amplification (as opposed to exponential amplification in PCR2) of the sequencing ladder, reducing the amount of template required for the sequencing reaction by a factor of the number of cycles used (typically up to 25- to 30-fold). We have previously reported the application of PCR and cycle sequencing methodologies to the large-scale sequencing of human heart cDNA libraries (2–4). Our protocol involved PCR amplification of cDNA inserts from l-phage plaque suspensions, followed by cycle sequencing of the PCR products using a nested, fluorescein-labeled primer. Recently, we explored the possibility of sequencing inserts directly from phage plaques. While linear amplification sequencing of single l plaques had been described using manual sequencing techniques (5), this approach has not been successfully generalized to the automated sequencing technologies used in most largescale sequencing projects, probably due to the currently lower sensitivity of these technologies. Other attempts to sequence very small quantities of template included coupled amplification and sequencing (6) and semiexponential cycle sequencing (SECS) (7). However, because both techniques are two-step processes, involving a PCR step followed by cycle sequencing of the PCR product, they have not yet been, in practice, particularly advantageous over more traditional template isolation and sequencing protocols currently used by large-scale sequencing projects. We therefore developed a method for single-step sequencing directly from individual l-phage plaques by exponential amplification (EAS). In this report, we present results of EAS performed on single plaques from a directionally cloned human fetal heart cDNA library, and we demonstrate its potential utility to large-scale sequencing projects.
Exponential amplification sequencing. A human fetal heart (10–12 weeks) l ZAP-Express cDNA library was constructed and plated using XL1-Blue as a host strain, using isopropyl b-D-thiogalactoside/X-gal color selection. Individual plaques were picked with glass Pasteur pipets and each was eluted in 100 ml SM buffer, as previously described (2). EAS reactions were conducted essentially according to the manufacturer’s recommended protocol using the Taq Cyclist DNA Sequenc2 Abbreviations used: PCR, polymerase chain reaction; SECS, semiexponential cycle sequencing; EAS, exponential amplification sequencing; EST, expressed sequence tag.
231, 460–463 (1995)
0003-2697/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ m4314$$nat
10-13-95 16:04:50
abnt
AP-Anal Bio
NOTES & TIPS
ing Kit (Stratagene). Beyond the buffer and nucleotide components provided by the kit (10 mM Tris–HCl, pH 8.8, 50 mM KCl, 0.001% gelatin, 4 mM MgCl2 , 2 mM dATP, 5 mM dCTP, 5 mM dGTP, 5 mM dTTP), the EAS master mix contained 10 pmol fluorescein-labeled modified T3 primer (5*-fluorescein-GAAATTAACCCTCACTAAAGGG-3*), 10 pmol unlabeled modified T7 primer (5*-CCAGTGAATTGTAATACGACTCACTATCGGGCG-3*), an additional 312.5 pmol each dNTP (1 ml 1 312.5 mM), 2 ml dimethyl sulfoxide, 1 ml phage suspension, and 5 U Taq, in a final total volume of 30 ml. Master mix (7 ml) was aliquoted into each of four tubes containing, respectively, 3 ml ddATP (600 mM), ddCTP (600 mM), ddGTP (100 mM), or ddTTP (1000 mM) (manufacturer’s concentrations). Reactions were overlaid with mineral oil and heated at 947C for 2 min, followed by 35 cycles at 947C, 30 s; 507C, 25 s; and 727C, 1 min 20 s; followed by a 5-min extension at 727C. Reactions were stopped by addition of 5 ml stop solution (95% formamide, 20 mM EDTA, 10 mg/ml blue dextran). Prior to electrophoresis, samples were heated to 957C. Samples were electrophoresed through a 6% acrylamide gel, using the Pharmacia A.L.F. DNA Sequencer. Data handling. Sequence comparisons were performed using the BLAST server at the National Center for Biotechnology Information (8,9) and the HIBIO package (Hitachi). Results and Discussion Table 1 summarizes the results of direct sequencing from individual plaques from a human fetal heart l ZAP-Express cDNA library by EAS. Sequences ranged in length from 142 to 503 bp, with the average length being approximately 296 bp. Sequencing reactions which were conducted in the absence of additional dNTP or unlabeled primer, or both, failed to generate detectable sequences (data not shown). Of 49 partial cDNA sequences, or expressed sequence tags (ESTs) obtained, 15 demonstrated no homology to known genes, while 3 exhibited similarity to other human ESTs, though not to any other known genes (Table 1). The remaining 31 were similar to previously known genes, including 2 novel human homologs of genes characterized in the cow. Of the 31 known ESTs, 7 represented mitochondrial transcripts (23%), while another 7 represented ribosomal proteins (23%). Assessment of sequencing accuracy by comparison of EST sequences with known gene sequences found that most known ESTs demonstrated between 98 and 100% identity to known gene sequences (Table 1), suggesting a high level of accuracy. The 296-bp average sequence length reported here exceeds the 200 bp previously reported for manual linear amplification sequencing from a single l-phage
/ m4314$$nat
10-13-95 16:04:50
abnt
461
plaque (5). Moreover, while the previous protocol used 80% of the phage plaque suspension, the EAS protocol described in this report used only 1% (i.e., 1 of 100 ml) of phage suspensions. This represents at least an 80fold decrease in the amount of template required for sequence generation or, conversely, an 80-fold increase in sensitivity over conventional cycle sequencing. Assuming the average l-phage plaque to contain 106 – 107 phage particles (10), and assuming each particle to represent one copy of the phage genome, this represents only 104 –105 copies, an unprecedented sub-attomole quantity of template required to generate quality sequence. Titering of phage suspensions demonstrated as few as 4 1 103 pfu required for sequence generation and detection (data not shown). Assuming similar requirements for template quantity in direct sequencing of genomic DNA, this method represents a requirement of only 0.02–0.4 mg of genomic DNA in order to sequence single-copy genes and would theoretically permit direct sequencing from genomic DNA isolated from less than a single drop of blood. Given the currently lower sensitivity of automated sequencing than manual techniques, it is reasonable to assume that the template requirements for manual sequencing protocols should be even lower. The rationale underlying the EAS technique is simple and similar to that of SECS (7). Standard cycle sequencing reactions (containing an excess of a fluorescein-labeled sequencing primer) were performed in the presence of additional nucleotides (dNTPs) and of an additional, unlabeled PCR primer, opposite in sense to the sequencing primer. The presence of additional dNTPs decreases the frequency of early termination by ddNTP and increases the probability of full-length extension from one primer to the annealing site of the other primer. Thus, in each cycle, a proportion of primer extensions from each primer would extend the full length of the fragment to be sequenced, thereby allowing for exponential amplification of the template. Concurrently, termination of the remainder of primer extensions by ddNTP leads to generation of a sequencing ladder. In early stages of cycling, when relatively more dNTP is available, proportionally more exponential amplification of the template occurs. In later stages, however, as dNTP becomes depleted, and as the ratio of dNTP/ ddNTP decreases, proportionally more ddNTP is incorporated, generating shorter fragments, thereby progressively diminishing the rate of exponential amplification, while progressively increasing the rate of linear amplification sequencing. In essence, EAS merges the concepts of PCR and linear amplification sequencing into a single step. It should be noted at this point that although sequencing ladders would be generated from both primers, only the ladder originating from the labeled primer would be visualized. To date, EAS has been successfully applied only to
AP-Anal Bio
462
NOTES & TIPS TABLE 1
Partial Sequences of cDNA Clones from a Human Fetal Heart cDNA Library by EAS GB/EMBL/DDBJ Accession Nos. R56917–R56964
Clone
Sequence length (bp)
Putative identity
EAS-J2362 EAS-J2376 EAS-J2390 EAS-J2396 EAS-J2403 EAS-J2420 EAS-J2434 EAS-J2491 EAS-J2499 EAS-J2517 EAS-J2765 EAS-J3574 EAS-J3596 EAS-J3606 EAS-J3608 EAS-J2782 EAS-J2757 EAS-J2346 EAS-J2374 EAS-J2356 EAS-J2363 EAS-J2767 EAS-J2525 EAS-J3590 EAS-J3611 EAS-J2383 EAS-J1323 EAS-J3610 EAS-J2436 EAS-J2500 EAS-J2364 EAS-J2768 EAS-J1319 EAS-J1322 EAS-J2392 EAS-J2424 EAS-J2774 EAS-J3582 EAS-J3613 EAS-J2775 EAS-J2412 EAS-J2761 EAS-J1324 EAS-J3588 EAS-J3609 EAS-J3594 EAS-J2780 EAS-J2400 EAS-J2433
142 307 400 328 267 279 211 295 266 262 338 165 241 336 299 503 412 322 323 360 330 206 317 227 233 337 333 331 188 246 222 207 302 337 412 301 291 208 312 259 243 351 450 165 287 358 334 407 277
Novel Novel Novel Novel Novel Novel Novel Novel Novel Novel Novel Novel Novel Novel Novel EST hbc2678 EST HSB92G052 EST IB287, IB2438 a-Globin Alu repetitive element Alu repetitive element Cardiac a-myosin heavy chain CCAAT/enhancer-binding protein d Chloride channel (cow) Chorionic gonadotropin Cytochrome c oxidase subunit VIIc EDZ protein Elongation factor 1-g Endozepine F1F0 ATPase synthase domain g subunit (cow) Ki nuclear autoantigen Kinesin heavy chain Mitochondrial Mitochondrial Mitochondrial Mitochondrial Mitochondrial Mitochondrial Mitochondrial Myosin light chain Open reading frame Ribosomal protein Ribosomal protein L3 Ribosomal protein L23 Ribosomal protein L27a Ribosomal protein L31 Ribosomal protein L32 Ribosomal protein S11 Ubiquinol cytochrome c reductase
% match
Length of match
94 99 96 96
246 304 184 308
98 97 74 98 98 98 99 97 88 98 99 96 99 99 99 98 98 99 99 98 98 98 98 98 100 99 98 96
206 316 91 233 288 301 331 188 238 221 206 302 319 412 300 291 208 298 258 243 342 420 163 287 358 333 381 132
Note. All matches listed are with human sequences, unless otherwise denoted in parentheses.
the sequencing of relatively short cDNA inserts (up to 1.1 kb; data not shown), likely because longer templates have a lower probability of full-length extension at the dNTP/ddNTP ratio used in this report. It is therefore expected that the dNTP/ddNTP ratio will be
/ m4314$$nat
10-13-95 16:04:50
abnt
a critical factor in optimizing EAS conditions to permit sufficient amplification at longer insert lengths. Nevertheless, the implications and applications of EAS are potentially numerous. Generation of highquality sequences directly from phage plaques or from
AP-Anal Bio
NOTES & TIPS
single bacterial colonies would reduce time and costs required for template isolation prior to sequencing. EAS may therefore be of great interest to large-scale sequencing projects making use of dye-primer-based sequencing chemistries, for example, in the rapid sequencing of size-selected shotgun libraries. Moreover, the ability to sequence from very small quantities of genomic DNA for rapid genotype analysis might be of interest to the field of molecular diagnostics. Acknowledgments. We acknowledge the excellent assistance of H. Ma and X.-G. Zhao. This work was supported in part by the Medical Research Council of Canada and by the Heart and Stroke Foundation of Ontario. D.M.H. is supported by a Medical Research Council of Canada Studentship.
463
2. Liew, C. C. (1993) J. Mol. Cell. Cardiol. 25, 891–894. 3. Liew, C. C., Hwang, D. M., Fung, Y. W., Laurenssen, C., Cukerman, E., Tsui, S., and Lee, C. Y. (1994) Proc. Natl. Acad. Sci. USA 91, 10645–10649. 4. Hwang, D. M., Hwang, W. S., and Liew, C. C. (1994) J. Mol. Cell. Cardiol. 26, 1329–1333. 5. Krishnan, B. R., Blakesley, R. W., and Berg, D. E. (1991) Nucleic Acids Res. 19, 1153. 6. Ruano, G., and Kidd, K. K. (1991) Proc. Natl. Acad. Sci. USA 88, 2815–2819. 7. Sarkar, G., and Bolander, M. E. (1995) Nucleic Acids Res. 23, 1269–1270. 8. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403–410. 9. Gish, W., and States, D. J. (1993) Nature Genet. 3, 266–272.
REFERENCES 1. Saluz, H., and Jost, J. P. (1989) Proc. Natl. Acad. Sci. USA 86, 2602–2606.
/ m4314$$nat
10-13-95 16:04:50
abnt
10. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, pp. 2–63, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
AP-Anal Bio